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Hepatic expression of the rat S14 gene is markedly and rapidly induced in response to T3. Previously, three contiguous restriction fragments of the S14 gene with thyroid hormone response activity were mapped to a region 2.5-3.0 kilobases upstream from the start of transcription [Far Upstream Regulatory region (FUR)]. To further investigate the molecular basis of the thyroid hormonal control of S14 gene expression, we have mapped the functional TRE sequences in the FUR region of the S14 gene. In vitro translated thyroid hormone receptor (TR) and retinoid X receptor were used in the gel retardation assays to map receptor binding sites in the S14 gene. Three TR-binding sequences were identified in the FUR region of the S14 gene and designated: FUR10 (from -2718 to -2694), FUR11 (from -2632 to -2595), and FUR12 (from -2582 to -2558). Each binding site contains two or more elements related to the consensus monomer binding motif 5'-Pu-GGTCA. In FUR10 and FUR12, these motifs were arranged as direct repeats with 4 base pair spacing, while in FUR11 a more complex arrangement occurred. From mutagenesis experiments, all three TR-binding sequences in the S14 gene were found to play a role and synergize with each other in the responsiveness to T3. The importance of this functional synergy is also shown by the observations that at least two TR-binding sites are required for T3 induction in hepatocytes. In addition, synergy occurs between TR and additional regulatory sequences present in the FUR region and provides the maximal T3 response of the S14 gene.

Functional synergism between multiple thyroid hormone response elements regulates hepatic expression of the rat S14 gene.